Mutations of specific viral genes of oncolytic herpes simplex virus (oHSV)-1 have been shown to confer selectivity to cancer cells, which has enabled translational studies in humans. However, only a subset of cancer patients inoculated with oHSV-1 have shown objective response in phase 1 and 2 clinical trials. We have recently identified GBM lines that are resistant to oHSV mediated oncolysis. Based on our findings, we have created recombinant oHSV-TRAIL, an oHSV bearing a secretable pro-apoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and hypothesize that oHSV-TRAIL could be used as a cancer therapeutic to target a broad spectrum of resistant tumors in a mechanism based manner. In this proposal, we will screen a panel of glioma stem cells (GSC) isolated from patient derived primary GBM lines, for their sensitivity to oHSV and TRAIL and identify lines that are resistant to oHSV or both oHSV and TRAIL. Based on our preliminary studies on the downregulation of ERK, activation of JNK and p38 and subsequent up- regulation of caspases in oHSV and TRAIL resistant glioma cells treated with oHSV-TRAIL, we hypothesize that oHSV will target cell proliferation pathway and prime oHSV and/or TRAIL resistant established glioma cells and primary GSCs to TRAIL mediated apoptosis. The efficacy of oHSV-S-TRAIL will be evaluated in vitro and in mouse models created from identified lines and ultimately in resistant lines identified from screening a panel of freshly resected patient tumors. The incorporation of genetically engineered fluorescent (GFP and mCherry) and bioluminescent (firefly luciferase; Fluc and Renilla luciferase;Rluc) imaging markers into oHSV and glioma cells will allow us to follow delivery and spread of oHSV in vivo; changes in glioma volumes and invasion; and the efficacy of oHSV-TRAIL by in vivo bioluminescence imaging (BLI) and intravital microscopy (IVM). We anticipate that our findings will have a major contribution towards: 1) elucidating molecular mechanisms of how oHSV mediated oncolysis and TRAIL can function in concert to target heterogeneous glioma cells which are resistant to viral oncolysis and/or cytotoxic therapies; and 2) ultimately developing novel oHSV based therapies for patients with brain tumors.